【作者】 赵杰；

【导师】 康振生；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2004， 硕士

【摘要】 小麦条锈病是小麦生产上的一种重要病害。使用抗病品种是防治该病经济而有效的措施。然而,品种抗锈性丧失常导致小麦条锈病大发生,现已明确毒性变异是品种抗锈性丧失的主要原因。由于传统的毒性分析方法本身缺乏遗传标记,限制了对条锈菌群体进化和毒性变异机制的进一步认识,严重制约了小麦条锈菌群体生物学研究。目前,有关小麦条锈菌的毒性基因的分子标记尚未见报道,因此,利用DNA分子标记技术寻找小麦条锈菌毒性基因的特异性标记,在理论上和生产上均有重要的意义。本文拟采用RAPD分子标记技术,通过揭示小麦条锈菌水源11致病类型与Hybrid46致病类型的遗传多样性,寻找其特异性分子标记,这些标记很可能与毒性基因相关,从而为阐明病菌的毒性演化规律、开发更有效的病害防治方法及建立生理小种的快速分子检测体系奠定基础。本文取得了以下主要结果:对小麦条锈菌的RAPD反应条件进行了优化,建立了适于小麦条锈菌的RAPD反应体系。反应体系为:10×Reaction PCR Buffer 2.5 μl,MgCl2 2.0 mmol/L,dNTPs 0.15 mmol/L,引物 20 ng,模板DNA 40 ng,Taq酶 1U,ddH2O 补充至总体积25 μl。扩增程序为:94℃预变性5 min; 94℃, 30s, 36℃, 40s, 72℃, 90s, 45个循环;72℃延伸7 min。共选用了140条10碱基随机引物,分别对5个Hybrid46致病类型菌系和8个小麦条锈菌水源致病类型菌系进行了RAPD分析,结果获得了一个可能与小麦条锈菌Hybrid46致病类型毒性基因相关分子标记(HY-T1)、两个与水源11致病类型毒性基因相关的分子标记(Su-T1、Su-T2),三个片段(HY-T1、Su-T1、Su-T2)均克隆到pGEM-T easy vector上,测序结果表明HY-T1标记的DNA大小为1052 bp;Su-T1和Su-T2标记的DNA大小分别为554 bp和818 bp。序列同源性比较发现HY-T1第926～947处与人的受体蛋白基因第43356～43335处完全同源;Su-T1第336～357处与人的受体蛋白基因第47975～47996处完全同源;Su-T2第261～283处、第15～37处均与Ascochyta(壳二孢属)rabiei DNA处第98～120处完全同源,其潜在意义有待于进一步研究。采用通用引物ITS1(TCCGTAGGTGAACCTGCGG)与ITS4(TCCTCCGCTTATTGATATGC),对5个小麦条锈菌菌株、小麦秆锈菌和小麦叶锈菌rDNA的内转录区(ITS)进行PCR扩增,克隆、测序,从NCBI中搜索了一个已注册的小麦条锈菌(登录号:AY114292)序列,将其与参试菌株序列进行Alignment比较,绘制了菌株间亲缘关系系统树,Alignment分析结果表明:小麦条锈菌与小麦秆锈菌、小麦叶锈菌间的序列存在较大的差异。并根据序列差异性设计了一对小麦条锈菌的特异引物,以此引物对包括小麦条锈菌在内的7种小麦叶部病害病原进行了PCR检测,结果表明:在优化的反应体系下及扩增条件下此对引物只在小麦条锈菌为模板的扩增体系<WP=6>中扩出一条大小约为170 bp的扩增产物,为小麦条锈病的早期快速诊断及对病害的监测和预测预报有重要的应用价值。更多还原

【Abstract】 Wheat yellow (stripe) rust, caused by Puccinia striiformis f.sp. tritici, is one of the most important diseases of wheat. The current strategy for management of the disease is use of resistant cultivars. However, the resistance breakdown of wheat cultivars results in epidemic of the disease. It is clear that virulence mutant is major reason of resistance breakdown of wheat cultivars. Because the virulence analysis of P. striiformis f.sp. tritici is short of genetic markers , and hampered the further research to population evolution and virulence mutant of P. striiformis f.sp. tritici, and the population bioresearch of P. striiformis f.sp. tritici has been restricted. Up to date, reference about molecular markers related to virulence genes of P. striiformis f.sp. tritici has not been found. Therefore, it is very important and useful in theory and in practice to fins some specific markers related to striiformis f.sp. tritici.This paper is planning to open out the genetic diversity of pathotype Shuiyuan11 and pathotype Hybrid46 of P. striiformis f.sp. tritici using RAPD molecular marker technique. In order to clarify the virulence evolutional mechanism of P. striiformis f.sp. tritici , and to search after effective control method of wheat rust disease, and to establish a practical molecular identification system of P. striiformis f.sp. tritici, specific molecular markers of two pathotypes were searched on a large scale. The results obtained as follows:1. An optimized RAPD analysis system for the pathogen was developed in this paper, which including: 10×Reaction PCR Buffer 2.5 μl, MgCl2 2.0 mmol/L, dNTPs 0.15 mmol/L, Primer 20 ng, DNA template 40 ng, Taq DNA polymerase 1U, add double distilled water up to 25 μl in the end. Amplification program: 5 min at 94℃ for initial denaturation, then 45 cycles that consisted of 30s at 94℃,40s at 36℃ and 90s at 72℃, followed by a final 7 min extension at 72℃.2. A total of 140 random primers analyzed in this paper. The RAPD analysis was conducted to find polymorphic fragments related to the virulence genes of pathotype Hybrid46 and pathotype Shuiyuan11 from five isolations of pathotype Hybrid46 and eight isolations of pathotype Shuiyuan11. Three specific markers were found, named by HY-T1, Su-T1 and Su-T2 respectively, thereinto, HY-T1 was probably related to the virulence gene virulence of pathotype Hybrid46 , and Su-T1,Su-T2 were that of pathotype Shuiyuan11. <WP=8>These three reproducible fragments were cloned into the pGEM-T easy vector. Three DNA sequences of showed that the length of HY-T1 was 1052 bp, that of Su-T1 was 554 bp, and that of Su-T2 was 818 bp. The results of homology analysis showed that one hundred percent alignment homology between 926-947 nucleotide in HY-T1 and 43356-43335 nucleotide of human receptor protein; that of between 336-357 nucleotide in Su-T1 and 47975-47996 nucleotide of human receptor protein; and that of between 261-283 nucleotide, and between 15-37 nucleotide in Su-T2 and 98-120 nucleotide in Ascochyta rabiei. Its significance should be studied furthmore.3. The sequences of rDNA internal transcribed spacer (ITS) of Puccinia striiformis f.sp. tritici(PST), P. graminis f.sp. tritici (PGT) and P. recondita f.sp. tritici (PRT) were amplified by PCR using universal primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) , and were cloned , and were sequenced. The alignment of these pathogens tested and another pathogen from NCBI(registered No. AY114292)showed that great interspecific variation in rDNA ITS1-5.8s-ITS2 sequence region. Phylogenetic tree was obtained. Sequences of ITS regions of the ribosomal gene repeat were used to design and to synthesize primers specific for PST detection by a polymerase chain reaction (PCR). A about 170 bp product of PCR was obtained and used to detect PST among these pathogens., which could be used to develop a rapid PCR-based diagnose test to wheat yellow rust disease at early stage of P. sfriiformis f.sp. tritici infesting wheat, and could be applied for inspection and forecast.更多还原